Allogeneic hematopoietic cell transplantation (HCT) is a curative therapy for relapsed hematological malignances (i.e. leukemia) due to graft-versus-leukemia (GVL) activity mediated by alloreactive T cells. However, alloreactive T cells also mediate graft-versus-host disease (GVHD), which remains the major obstacle for wide-spread application of allogeneic HCT. The long-term goal of our study is to develop novel regimens that prevent GVHD while preserving GVL activity. PD-L1 interacts with CD80 and PD-1. Although PD-L1/PD-1 regulation of immune responses plays an important role in animal models and humans, the role of PD-L1/CD80 remains largely unknown. Our recent publication in JCI has shed light on the importance of PD- L1/CD80 interactions. Our studies indicate that the outcome of PD-L1-mediated signaling in CD8+ T cells depends on the presence or absence of CD4+ T cells, the nature of the interacting receptors (i.e. CD80 versus PD-1) expressed by CD8+ T cells and the tissue environment (i.e. lymphoid versus parenchymal tissues) in which the signaling occurs. We observed that, in the absence of donor CD4+ T cells, CD8+ T-T interactions via PD-L1/CD80 augmented nave/activating CD8+ T proliferation and survival in lymphoid tissues, leading to strong GVL activity. In contrast, host-tissue PD-L1 interactions with PD-1 and CD80 on CD8+ T cells in GVHD target tissues induced their proliferation and apoptosis, leading to prevention of GVHD. Donor CD4+ T cells helped CD8+ T cells via IL-2 become resistant against tissue PD-L1-mediated tolerance. Agonistic PD-L1-Ig binding to both CD80 and PD-1 augmented activated T cell proliferation and apoptosis. Conflicting results have been reported regarding the effects of glycolysis and oxidative phosphorylation (OXPHOS) in alloreactive T cells during the pathogenesis of acute GVHD. In kinetic studies, we observed that expression of CD80 and PD- 1 was associated with shift from glycolysis to OXPHOS. Therefore, we hypothesize that 1) PD-L1/CD80 and PD-L1/PD-1 interactions reciprocally regulate T cell glycolysis and OXPHOS, and the outcome depends on the tissue environment, due to differential T cell expression of CD80, PD-1, and PD-L1; 2) Reduction of serum IL-2 will increase the sensitivity of T cells towards tissue PD-L1-mediated or agonistic PD-L1- Ig-mediated tolerance in association with metabolic profile changes. The proposed studies will dissect the mechanisms by which PD-L1/CD80 interactions regulate glycolysis and OXPHOS in nave/activating and activated T cells in lymphoid and GVHD target tissues (Aim 1). We will also design a regimen of sequentially administered anti-IL-2 and agonistic PD-L1-Ig to prevent GVHD and preserve strong GVL activity (Aim 2). These studies will reveal novel insights into T cell biology and GVHD pathogenesis and could lead to development of novel regimens that prevent GVHD while preserving GVL activity in humans. 1